While a functional cytochrome P450-dependent mixed function oxidase system has been identified in the kidney, the distribution of isozymes along the tubule epithelium and their relative contribution to arachidonic acid metabolism has not been completely characterized. Products of P450-dependent arachidonic acid metabolism mediate natriuretic responses in the proximal tubule in response to angiotensin II (AII). The purpose of this proposal is to establish the identity of the isozymes responsible for arachidonic acid concentration and activity, focusing on the proximal convoluted tubule. The metabolic profile of each isozyme in arachidonic acid metabolism will be determined and compared with the profile from intact cells. The effect of induction of specific isozymes by treatment of rabbits with established inducers such as phenobarbital, B-naphthoflavone, and clofibrate on arachidonic acid metabolism in response to AII will be determined to try and establish the identity of the hydroxylase. These experiments will be complimented by inhibitors of different isozymes which may be present in proximal cells. If the arachidonic acid hydroxylase appears to be a unique P450 as a result of inhibitor and induction experiments, it will be isolated and characterized from kidney cortex microsomes. This will be accomplished by a combination of chromatofocusing and ion exchange HPLC, monitoring the fractions for hydroxylase activity. Having established the identity of the arachidonic acid hydroxylase, experiments will be run to determine those factors which regulate the expression of the enzyme by coupling in situ hybridization histochemical studies with treatments which alter AII and/or the P450 concentrations. Experiments will also be done to determine whether the hydroxylase is expressed and is inducible in tissue culture.

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National Heart, Lung, and Blood Institute (NHLBI)
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Case Western Reserve University
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